Cholesterol esterases with variable substrate specificity

ABSTRACT

For the preparation of cholesterol esterases with variable substrate specificity by cloning of a cholesterol esterase gene, the active center of which has the sequence -Gly-His-Ser-X-Gly-, wherein X signifies an amino acid, into a vector, transformation of a micro-organism with this vector and expression of the cholesterol esterase gene, one exchanges the amino acid X of the active center for another amino acid by mutagenesis.

The invention concerns a process for the preparation of cholesterolesterases with changed substrate specificity, with cholesterol esterasesobtained in this way, as well as with their use for the enzymaticdetermination of cholesterol.

The determination of cholesterol in serum is an important parameter inthe diagnosis of arteriosclerosis. The cholesterol present in serum ispresent in very heterogeneous compounds, whereby about 70 to 80% of thecholesterol is esterified with fatty acids of varying length. Thus, theexact determination of the cholesterol level depends upon how completelythe cholesterol esters present are cleaved to free cholesterol. Inclinical and medical practice, the enzymatic cleavage of the cholesterolesters by means of cholesterol esterase has proven to be the simplestprocess. However, it is a disadvantage of this determination thatdifferent cholesterol esters are cleaved by particular cholesterolesterases with differing specificity. Therefore, it is an object of theinvention to make available cholesterol esterases with changed substratespecificity and improved activity.

According to the invention, this task is solved by a process for thepreparation of cholesterol esterases with changed substrate specificityby cloning of a cholesterol esterase gene, the active site of which hasthe sequence -Gly-His-Ser-X-Gly-, wherein X signifies an amino acid,into a vector, transformation of a micro-organism with this vector andexpression of the cholesterol esterase gene, which is characterised inthat one exchanges the amino acid X of the active site for another aminoacid by mutagenesis.

The invention concerns cholesterol esterases which, as active site (i.e.as region responsible for the enzymatic activity) contains the aminoacid sequence -Gly-His-Ser-X-Gly-, whereby X signifies any desired aminoacid. Such cholesterol esterases with different amino acid residues Xare known (see e.g. Table 1). However, these cholesterol esterasesoriginate from different organisms and, consequently, also differ in thenucleotide sequence of the genes coding therefor. According to thepresent invention, a process is now made available for obtaining a batchof different cholesterol esterases from a single cholesterol esterase ina simple and rapid manner by mutagenesis of the amino acid X of theactive site, which only differ in the amino acid X. However, by thechange of the nucleotide sequences in the cholesterol esterase gene, atthe same time an additional change of the peptide frame outside of theactive site can also take place insofar as this is desired.

Surprisingly, it has been found that the substrate specificity ofcholesterol esterases, the active site of which has the amino acidsequence -Gly-His-Ser-X-Gly-, can be changed in that one exchanges theamino acid X in the active site by another amino acid. This isexpediently achieved by mutation of the codon which codes for the aminoacid X in the active site. The mutagenesis is preferably carried out inan objective manner, namely, with the use of oligonucleotides. One cutsout from the cholesterol esterase gene an oligonucleotide which containsthe coding region for the active site and, in place thereof, ligates anew oligonucleotide into the gene which, in the place of X, codes foranother amino acid.

The new oligonucleotide is preferably synthesised by chemical means. Thesynthesis is expediently carried out on a solid phase. Such solid phasemethods are described summarily in E.-L. Winnacker, Gene und Klone, VCHVerlagsgesellschaft Weinheim (1985), pages 44 et seq. The newoligonucleotide is expediently prepared according to the amiditeprocess, especially the phosphoramidite process.

Alternatively to the chemical synthesis, it has also proved to beexpedient to obtain the new oligonucleotide from genes or gene ofbuilding blocks of other lipase/cholesterol esterases. One usuallyproceeds in a manner such that one selects the gene from such alipase/cholesterol esterase which already displays the substratespecificity for the desired ester bond. From such a gene the nucleotidesequence coding for the active centre is cut out and is introduced intothe cholesterol esterase gene to be changed as new active centre.

In the process according to the invention, there is preferably used thecholesterol esterase gene which corresponds to the amino acid sequenceaccording to SEQ ID NO:1.

In the process according to the invention, it has also proven to beexpedient that, for the transformation and for the expression of thecholesterol esterase gene, a micro-organism is used which itself doesnot express an active cholesterol esterase. As such a micro-organism,there is particularly used a Pseudomonas or E. coli strain, especiallypreferably Pseudomonas spec. DSM 5902. Micro-organisms which do notexpress a cholesterol esterase can be produced by by mutagenizing astrain which produces cholesterol esterase so that it no longerexpresses active cholesterol esterase. This strain is then transformedwith a vector (e.g. a plasmid) which carries the gene for thecholesterol esterase with changed substrate specificity.

However, it is also possible to carry out the process according to theinvention with a strain of a micro-organism which expresses cholesterolesterase. In comparison with the original strain, such a strain willthen display increased expression. In the process according to theinvention, for this purpose there are preferably used, as startingstrains, E. coli or Pseudomonas strains.

According to the invention, as vectors for the cholesterol esterasegene, there are preferably used pBR322, pUC18 or pBT306.1 (EP-A-0 187138).

In the process according to the invention, as codon for the variableamino acid X, there is preferably used, in each case, the codon which isalso present in the corresponding micro-organism or vector. However, ithas proved to be especially expedient to use as codon for X, for HisCAT, for Met ATG, for Phe TTC, for Gln CAG, for Leu TTG, for Ile ATC,for Val GTC, for Ser TCT, for Pro CCC, for Thr ACT, for Ala GCT, for TyrTAT, for His CAC, for Asn AAC, for Lys AAG, for Asp GAC, for Glu GAG,for Cys TGT, for Arg CGT and for Gly GGA.

The amino acid introduced by the mutation preferably is Leu, Gln, Met orHis. The amino acid Gln is especially preferred.

The invention also concerns new, hitherto unknown cholesterol esteraseswhich are obtainable via the process according to the invention. Inparticular, however, the invention concerns a mixture of thosecholesterol esterases obtainable by the process according to theinvention which cleave every cholesterol ester bond. According to theinvention, this can be achieved in that one produces cholesterolesterases which contain in the active centre all 20 amino acids possiblefor X. With a mixture of such cholesterol esterases according to theinvention, preferably a mixture of all 20 different cholesterolesterases, it is possible to completely cleave in a simple mannercompletely the various cholesterol esters occurring in the body.

Therefore, the invention also concerns the use of cholesterol esterasesobtained in this way for the determination of cholesterol in bodyfluids, as well as a reagent which contains these or a mixture thereof.

The strain Pseudomonas spec. DSM 5902 and the plasmid pBTmglCE with thedeposit number DSM 5903 were deposited on the 24th Apr., 1990, at theDeutsche Sammlung von Mikroorganismen (DSM) (German Collection ofmicro-organisms), Mascheroder Weg lb, D-3300 Braunschweig. TheEscherichia coli strain HB 1254 with the deposit number DSM 6541, hasbeen deposited on the 29.05.1991 at the Deutsche Sammlung vonMikroorganismen.

The invention is explained in more detail by the following Examples, inconjunction with the sequence protocols.

SEQ ID NO:1 shows the amino acid sequence of the cholesterol esterase ofPseudomonas spec.

SEQ ID NO:2 and 3 show 2 complementary oligonucleotides which togethergive an EcoRI/SalI fragment which codes for amino acids 1 to 30 of themature cholesterol esterase.

SEQ ID NO:4 and 5 show the oligonucleotides used for the fusion of thecloned cholesterol esterase to the GBP signal peptide via a deletionmutagenesis.

EXAMPLE 1 Cloning of the Pseudomonas spec. gene which codes forcholesterol esterase (CE)

DNA was isolated from a micro-organism of the genus Pseudomonas,digested with suitable restriction enzymes and cloned into a vectorcompatible for E. coli, such as pBR 322. The identification ofCE-containing plasmids took place via oligonucleotides which werederived from the peptide sequence of the Pseudomonas spec. cholesterolesterase. With the use of oligonucleotide samples which were derivedfrom the amino terminus of the CE protein, there was isolated a 2.1kb-sized PstI fragment of the Pseudomonas spec. DNA which was thensubcloned into pBR322. DNA sequencing showed that this fragment onlycoded the N-terminal part of the cholesterol esterase. With the help ofthe PstI fragment as sample, the C-terminal part of the CE-coding DNAwas identified on an approximately 1.1 kb-sized SalI fragment and alsosubcloned into pBR322.

The amino acid sequence of the overlapping PstI and SalI fragmentsderived from the DNA gave an open reading frame of 316 amino acids. Themolecular weight of the mature protein is 30770 D.

Cholesterol esterase is an enzyme localised in the periplasma or theouter membrane. A signal peptide of 24 amino acids is responsible forits secretion. The protein sequences, which were found by sequencingvarious preparations of the enzyme isolated from Pseudomonas spec. andpurified, are themselves not homogeneous. At the +1 position of themature cholesterol esterase either the amino acid Phe or Trp (seeSEQ.ID.NO:1) can be positioned, whereby there is given a signal peptidecleavage position is located between amino acid 24 and 25 or betweenamino acid 25 and 26. Therefore, in the case of expression in E. coli,account is to be taken of the fact that the N-terminus can be variable.

Signal peptide cleavage positions should be used which are recognized inE. coli (Van Heijne, Eur. J. Biochem., 133 (1983), 17-21; Van Heijne, J.Mol. Biol., 184 (1985), 99-105). The vector M13mglEcoK (cf. WO 88/09373)contains such a signal peptide cleavage position. This vector containsan about 700 bp-sized EcoRI/BamHI DNA fragment from the mgl operon fromS. typhimurium (Benner-Luger and Boos, Mol. Gen. Genet., 214 (1988),579-587) which contains not only the promoter but also the translationinitiation region and the signal peptide of the galactose bindingprotein (GBP). The cleavage position of this signal peptide isfrequently used in E. coli (Van Heijne, Eur. J. Biochem., 133 (1983),17-21).

For the completion of the C-terminal 1.1 kb-sized SalI fragment of thecholesterol esterase, two complementary oligonucleotides (seeSEQ.ID.NO.:2 and 3, corresponding to the amino acid Phe on position +1up to the amino acid Val in position +30) were inserted into the vactorpUC18, whereby pUC18* resulted. The 1.1 kb-sized SalI fragment of thecholesterol esterase sub-cloned into pBR322 was cloned into pUC18* whichhad been linearized with SalI. The plasmid pCHE1 thereby results.

For the fusion to the GBP signal peptide, the cholesterol esterase genecloned into pCHE1 was subcloned into the vector M13mglEcoK. For thispurpose, the vector M13mglEcoK was cleaved with KpnI, the overlapping3'-end digested off with T4 polymerase and subsequently cleaved withSalI. Into the so-modified vector was inserted the approximately 280 bplong N-terminal PvuII/SalI fragment of the cholesterol esterase frompCHE1. The vector M13mglEcoKCE resulted. Fusion of the maturecholesterol esterase to the signal peptide of the GBP took place viadeletion mutagenesis on the single-stranded DNA of M13mglEcoKCE. Byhybridisation with an oligonucleotide (SEQ.ID.NO.:4 or SEQ.ID.NO.:5),there results a partial heteroduplex DNA which is made up in vitro tothe double strand and transfected into E. coli HB2154 (Carter et al.,Nucl. Acids. Res., 13 (1985), 4431-4443). Such DNA molecules, which alsocontain additional sequences (GBP, EcoK cassette and 5'-untranslatedcholesterol esterase sequence) between the signal peptide sequence andthe mature cholesterol esterase sequence, were eliminated via EcoKselection in E. coli HB2154 (DSM 6541). The M13mglCE clones obtainedwere characterized via restriction analysis and DNA sequencing. By meansof the oligonucleotides used, two different M13mglCE derivativesresulted. These were cleaved with EcoRI and XhoI and the insertion(consisting of mgl promoter, signal peptide and N-terminal region of thecholesterol esterase) cloned back into the vector pCHE1 cleaved with theappropriate enzymes. A plasmid is obtained which contains the completesequence of the mature cholesterol esterase fused to the GBP signalpeptide sequence when the oligonucleotide SEQ.ID.NO.: 4 was used for thedeletion mutagenesis. In the case of the use of the oligonucleotideSEQ.ID.NO.:5 for the deletion mutagenesis, the plasmid pBTmglCE (DSM5903), which contains the amino acid Trp in position +1 of the maturecholesterol esterase sequence is obtained.

EXAMPLE 2 Changing of the Substrate Specificity by Modification of theDNA Sequence Coding for the Active Site

The active centre of cholesterol esterase/lipase is defined by the aminoacid sequence -Gly-His-Ser-X-Gly. This sequence, including the signalpeptide, is given in the case of the Pseudomonasspec.-lipase/cholesterol esterase by amino acids 109 to 113 with thesequence Gly, His, Ser, His, Gly. The active centre of a selection oflipases and cholesterol esterases is shown in Table 1.

The specificity of the cholesterol esterase can be changed by objectiveexchange of the amino acid X in the sequence Gly-His-Ser-X-Gly.

The sequence of the CE-coding DNA, which codes for amino acids 109-115(including the signal sequence), reads:

    Gly-His-Ser-His-Gly-Gly-Pro 5'-GGC GAC AGC CAT GGC GGC CCG-3'

The corresponding counter-strand reads:

    5'-CGG GCC GCC ATG GCT GTC GCC-3'

The following nineteen oligonucleotides are used for directedmutagenesis: ##STR1##

The mutagenesis is carried out according to known techniques on the M13template (Amersham No.1523 "Oligonucleotide-directed in vitromutagenesis system").

For this purpose, an approximately 970 bp-long SalI fragment of theCE-coding DNA from pBTmglCE is ligated into the double-stranded form ofthe phage DNA M13mpl9 which has been cleaved with SalI. Afterpreparation of single-strand DNA, the corresponding oligonucleotide ishybridized on the single-strand DNA and elongation carried out in the5'→3' direction beyond the oligonucleotide with the use of Klenowpolymerase, ligase and the four nucleotide triphosphates GTP, CTP, TTPand ATP. The now double-stranded DNA is transformed in E. coli (fromAmersham kit No. 1523 or from the mutagenesis kit of Boehringer MannheimGmbH, cat. No. 1269046). Individual plaques are picked and the M13phages contained therein are used for the preparation of single-strandDNA. DNA sequencing is carried out according to known techniques andthus the exact exchange to the desired mutation tested for. Afterpreparation of double-strand DNA, the approximately 970 bp-long mutatedSalI fragment is isolated. This fragment is then cloned back into thevector fragment of the plasmid pBTmglCE cleaved with SalI.

                  TABLE 1                                                         ______________________________________                                                         sequence of the active                                       lipases/esterases from                                                                         centre                                                       ______________________________________                                        hog pancreas     Gly--His--Ser--Leu--Gly                                      rat tongue       Gly--His--Ser--Gln--Gly                                      Staphylococcus   Gly--His--Ser--Met--Gly                                      Pseudomonas fragi                                                                              Gly--His--Ser--Gln--Gly                                      Pseudomonas spec.                                                                              Gly--His--Ser--His--Gly                                      gastric lipase   Gly--His--Ser--Gln--Gly                                      ______________________________________                                    

EXAMPLE 3 Expression of Lipase/Cholesterol Esterase from Pseudomonas

Plasmid pBTmglCE is cleaved with NaeI. The SacI linker d(CGTCGACG) isligated into this cleavage position. After preparation of DNA, thesuccessful ligation of the SacI linker is tested for by cleavage withSacI and an approximately 1800 bp-long fragment is isolated aftercleavage with EcoRI and SacI. Vector pBT306.1 (preparation according toEP-A-0 187 138) is cleaved with EcoRI and SacI. The approximately 1800bp-long fragment is ligated into this vector. The so-resulting plasmidbears the designation pBT306CE_(His) (wt).

This plasmid is transferred by in vivo transfer (Gene, 16 (1981),237-247) into the Pseudomonas mutant, DSM 5902. This strain ischaracterised by the fact that it does not code any active cholesterolesterase. Table 2 shows the expression in various Pseudomonas strains.The determination of activity took place as described in Example 5.

EXAMPLE 4 Culturing of Micro-Organisms which Contain Cloned CholesterolEsterase on a Plasmid

The culturing of the strains takes place without inductor at 30° C.overnight in a medium which contains 16 g bactotrypton (Difco), 10 gyeast extract (Difco) and 5 g sodium chloride per liter.

Table 2 shows the cholesterol esterase activity obtained.

                  TABLE 2                                                         ______________________________________                                                     with plasmid                                                     Pseudomonas  pBT306.1   with plasmid                                          strain       (control)  pBT306CH.sub.His (wt)                                 ______________________________________                                        wild type P  25*        130                                                   DSM 5902     0          120                                                   Pseudomonas  0          118                                                   alcaligenes                                                                   ______________________________________                                         *Units/liter/OD of the culture at 550 nm with cholesterol linoleate as        substrate (average of 5 independent measurements, determination to Exampl     6).                                                                      

EXAMPLE 5 Change of the substrate specificity, activity determination

Various derivatives of cholesterol esterase were prepared as describedin Example 2 by exchange of the amino acid X in the active centre.Exchange of the amino acid 112 His for glutamine leads to an increase ofthe activity towards the substrate cholesteryl-3-glutaric acid resorufinester (esterase colour substrate, Example 5b).

                  TABLE 3                                                         ______________________________________                                                        enzyme activity with CE in                                                    which amino acid 112 is:                                      substrate         His       Gln                                               ______________________________________                                        lipase color substrate                                                                          13.7      13.0                                              (Example 5a)                                                                  esterase colour   4.5       6.2                                               substrate (Example 5b)                                                        substrate in Example 5c:                                                      cholesteryl linoleate                                                                           120       93                                                cholesteryl octanoate                                                                           103       59                                                cholesteryl n-butyrate                                                                          8.3       4.1                                               cholesteryl oleate                                                                              210       113.3                                             ______________________________________                                    

As can be seen from Table 3, these esterases show very different enzymeactivities with respect to different substrates. This can beadvantageous in the case of the reaction of substrate mixtures.

a) Lipase determination (process 1)

For the determination of lipase, 1,2-0-dilauryl-rac-glycero-3-glutaricacid resorufin ester is cleaved with lipase. The resulting glutaric acidresorufin ester hydrolyses to glutaric acid and resorufin. Theextinction of resorufin is measured at 572 nm.

Reagents:

1. Substrate solution

1 mg 1,2-0-dilauryl-rac-glycero-3-glutaric acid resorufin esterdissolved in 1 ml dioxane/Thesit® (Boehringer Mannheim, No. 836630)(1:1).

2. Buffer solution

0.1 mol/l potassium phosphate buffer, pH 6.8.

3. Sample solution

6-8 U lipase/l in potassium phosphate buffer, pH 6.8.

For the carrying out of the determination, 0.85 ml of buffer solutionand 0.1 ml of substrate solution are mixed, warmed to 25° C. and thereaction started with 0.05 ml of sample solution. The extinction changeat 572 nm is monitored and ΔE/min. calculated from the linear range.

The lipase activity is calculated according to the following equation:##EQU1## b) Cholesterol esterase determination (process 2).

In the case of this process, the substrate cholesteryl-3-glutaric acidresorufin ester is converted with cholesterol esterase into cholesteroland glutaric acid resorufin ester. Glutaric acid resorufin esterhydrolyzes to resorufin, the color of which is measured at 572 nm.

Reagents:

1. Substrate solution

2 mg of substrate cholesteryl-3-glutaric acid resorufin ester dissolvedin 1 ml dioxane/Thesit® (1:1) and 30 μl acetic acid (2 mol/l ).

2. Buffer solution

0.1 mol/l potassium phosphate buffer, pH 6.8.

3. Sample solution

3-5 U cholesterol esterase/l diluted with potassium phosphate buffer, pH6.8.

For carrying out the determination, 0.85 ml of buffer solution and 0.05ml of substrate solution are mixed, warmed to 25° C. and the reactionstarted with 0.1 ml of sample solution. The extinction change at 572 nmis monitored and ΔE/min calculated from the linear range.

The cholesterol esterase activity is determined according to theequation: ##EQU2##

V=test volume (1 ml)

V=sample volume (0.1 ml)

ε=extinction coefficient of resorufin at 572 nm and at pH 6.8 (60.0[1·mmole⁻¹ ·cm⁻¹ ])

d=layer thickness of the cuvette (1 cm)

ΔE/min=extinction change/min.

c) Determination of cholesterol (process 3)

According to this process, the substrate is cleaved by cholesterolesterase into cholesterol and fatty acid, the resultant cholesterol isconverted with cholesterol oxidase into cholestenone and hydrogenperoxide and the resultant hydrogen peroxide is reacted with4-aminoantipyrine and phenol to give a coloured material, the colour ofwhich is measured at 500 nm on a photometer.

Reagents:

1. Reaction mixture

0.25 mol/l potassium phosphate buffer, pH 7.0

5 mg/ml sodium cholate

2.4 mg/ml phenol

0.4 mg/ml 4-aminoantipyrine

2. Peroxidase solution

6000 U/ml peroxidase (activity about 100 U/mg) in 0.25 mol/l potassiumphosphate buffer, pH 7.0.

3. Cholesterol oxidase

25 U/ml cholesterol oxidase (activity 25 U/mg) in 1 mol/l NaCl.

4. Substrate solution

2 μmol/l of one of the following substrates:

cholesteryl linoleate

cholesteryl octanoate

cholesteryl n-butyrate

cholesteryl oleate.

For carrying out the determination, 2.5 ml of reaction mixture, 0.01 mlperoxidase solution, 0.2 ml cholesterol oxidase solution and 0.5 mlsubstrate solution are mixed, warmed to 37° C. and the reaction startedwith 0.05 ml of sample. The extinction change is monitored, ΔE/min.calculated from the linear range.

The cholesterol esterase activity is calculated as follows: ##EQU3##

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1721 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Pseudomonas spec.                                               (xi ) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                      ACGCCAACCAGCGCTTAGCGAATTCAGCGCTGCAAACAACACCCTGTGCGCCCCGCAAGT60                AGCTGTTTAAATCGGCGCAGGGCGCATCCTAGAGACTCCCACGCAGGACTGTCAAGGCGC120               GCCATCATCGCACCCCAACCTTCGATTGGCCGCCTCAC ATTTCCACCGCTGGACGCGGGT180              CAAACGCTCCGCCTGCACTCGTCAAACCGCTGCCGAGCTGAACGATTGCGGCCTGCCATC240               ACAGCTCGGCTGTGTCGCCACGTAACAGAAAAGCTGCCGCAATCTCGCTGACAAAACAGG300               GTTATCTTCGGGAAA GCGCCGCAATAGAGGGGTTAATCGGGAAAACCTGGCGATGGCATG360              TTTCCTGCCATCGTTCTGTCGCCGTACGTGCCATGGGATGGGCACGCTTGTGTTGCAGGA420               TGCAGCCTGACTGCTAATAAACCCATCGAGAGCGCCATGAAGAACAATAAAACC 474                    MetLysAsnAsnLysThr                                                            24-20                                                                         CTGCTCGCCCTCTGCCTCGGCGCCGGCCTGCTCGCCAGCGGCCAGACC 522                          LeuLeuAlaLeuCysLeuGlyAlaGlyLeuLeuAlaSerGlyGlnThr                              15-10-5                                                                       CAGGCTTTCTGGTTCGGTTCGTCCGGCTATACCCAGACCAAATACCCC 570                          GlnAlaPheTrpPheGlySerSerGlyTyrThrGlnThrLysTyrPro                              1510                                                                          ATCGTCCTCGGCCACGGCATGCTGGGTTTCGACAGCATCCTCGGCGTC618                           Ile ValLeuGlyHisGlyMetLeuGlyPheAspSerIleLeuGlyVal                             15202530                                                                      GACTACTGGTATGGCATCCCGACCGCTCTACGCCGCGACGGCGCCAGC666                            AspTyrTrpTyrGlyIleProThrAlaLeuArgArgAspGlyAlaSer                             354045                                                                        GTCTACGTGACCGAAGTCAGCCAGTTGGACACCTCTGAAGCACGCGGC714                           ValTyrValThrGluValSerGlnLeuAspThrSerGluAlaArgGly                              505560                                                                        GAACAATTGCTGCAGCAGGTAGAGGACATCGTCGCCATCAGCGGCAAG762                           G luGlnLeuLeuGlnGlnValGluAspIleValAlaIleSerGlyLys                             657075                                                                        GGCAAGGTCAATCTGATCGGCCACAGCCATGGCGGCCCGACCACCCGC810                           GlyLys ValAsnLeuIleGlyHisSerHisGlyGlyProThrThrArg                             808590                                                                        TATGTCGCCGCCGTGCGCCCGGATCTGGTCGCTTCGGTCACCAGCGTC858                           TyrValAlaAlaVal ArgProAspLeuValAlaSerValThrSerVal                             95100105110                                                                   GGCGCTCCGCACAAGGGTTCGGCCACTGCAGACTTCCTCAAGGGCATC906                           GlyAlaProHi sLysGlySerAlaThrAlaAspPheLeuLysGlyIle                             115120125                                                                     AGCGACGGCCCTGCCGGGCCGGTAGCGACCCCGGTGCTGGCAGGCATC954                           SerAspGlyP roAlaGlyProValAlaThrProValLeuAlaGlyIle                             130135140                                                                     ATCAACGGCCTGGGCGCGCTGATCAACTTCCTCTCCGGCAGCCCCAGC1002                          IleAsnGlyLeu GlyAlaLeuIleAsnPheLeuSerGlySerProSer                             145150155                                                                     ACCACACCGCAGAACGCGCTCGGCTCGCTGGAGTCGCTCAACAGTCAA1050                          ThrThrProGlnAsnAla LeuGlySerLeuGluSerLeuAsnSerGln                             160165170                                                                     GGTGCCGCTCGCTTCAACGCCAAGTTCCCGCAGGGCATCCCGACCAGC1098                          GlyAlaAlaArgPheAsnAlaLysPh eProGlnGlyIleProThrSer                             175180185190                                                                  GCCTGCGGCGAAGGCGCCTACAGCGTGAACGGCGTGCGTTACTACTCG1146                          AlaCysGlyGluGlyAlaTyrS erValAsnGlyValArgTyrTyrSer                             195200205                                                                     TGGAGCGGCACCAGCCCGTTGACCAACCTGCTCGACCCGAGCGACCTG1194                          TrpSerGlyThrSerProLeu ThrAsnLeuLeuAspProSerAspLeu                             210215220                                                                     CTGATGGGCGCGTCCTCGTTGACCTTCGGCAGCGAAGCCAACGAGCCT1242                          LeuMetGlyAlaSerSerLeuThr PheGlySerGluAlaAsnGluPro                             225230235                                                                     GGTCGGCCGCTGCAGTTCGCGCATGGGCCAGTCATTCGTGACAACTAC1290                          GlyArgProLeuGlnPheAlaHisGlyPr oValIleArgAspAsnTyr                             240245250                                                                     CGGATGAACCACCTCGACGAGGTCAACCAGACGCTGGGGCTGACCAGC1338                          ArgMetAsnHisLeuAspGluValAsnGlnThrLeuG lyLeuThrSer                             255260265270                                                                  CTGTTCGAGACCGACCCGGTGACCGTCTACCGTCAACACGCCAACCGC1386                          LeuPheGluThrAspProValThrValTyrArg GlnHisAlaAsnArg                             275280285                                                                     CTGAAAAACGCCGGGCTCTAGGCTAACGGATAACTTCCGCCCGAGCCG1434                          LeuLysAsnAlaGlyLeu                                                            290                                                                            TGCGCATCGAGACATCGGTGCACGGCGCATCCTACCCCTGACATCCAGAGCCACACGTGA1494             AGAAAGCCCTATTCGCCCTGCCTCTACTGATCGGCGCCGGCCTGGCGTTGATGCTTTACC1554              TGCAACCCGGACATCAGCCCACCCACGTCAGCTCTCCTGCCACGGC TACAGTGACGAAAC1614             CTGTGCCGCAGGCACCTGCCGAAGCCATGACGCCGGCTGCAAGTGACACGCAGAAGAAGG1674              CGCCCAAACTGGCCCTGCCCGCCTCCTTCGCCGGCACCGACGTCGAC1721                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 316 amino acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetLysAsnAsnLysThrLeuLeuAlaLeuCysLeuGlyAlaGlyLeu                              24- 20-15 -10                                                                 LeuAlaSerGlyGlnThrGlnAlaPheTrpPheGlySerSerGlyTyr                              515                                                                           ThrGlnThrLysTyrProIleValLeuGlyHisGlyMetLeuGlyPhe                               101520                                                                       AspSerIleLeuGlyValAspTyrTrpTyrGlyIleProThrAlaLeu                              25303540                                                                      ArgArgAspGlyAlaSer ValTyrValThrGluValSerGlnLeuAsp                             455055                                                                        ThrSerGluAlaArgGlyGluGlnLeuLeuGlnGlnValGluAspIle                              60 6570                                                                       ValAlaIleSerGlyLysGlyLysValAsnLeuIleGlyHisSerHis                              758085                                                                        GlyGlyProThrThrArgTyrValAlaAlaValArgProAs pLeuVal                             9095100                                                                       AlaSerValThrSerValGlyAlaProHisLysGlySerAlaThrAla                              105110115120                                                                  AspPhe LeuLysGlyIleSerAspGlyProAlaGlyProValAlaThr                             125130135                                                                     ProValLeuAlaGlyIleIleAsnGlyLeuGlyAlaLeuIleAsnPhe                              140 145150                                                                    LeuSerGlySerProSerThrThrProGlnAsnAlaLeuGlySerLeu                              155160165                                                                     GluSerLeuAsnSerGlnGlyAlaAlaArg PheAsnAlaLysPhePro                             170175180                                                                     GlnGlyIleProThrSerAlaCysGlyGluGlyAlaTyrSerValAsn                              185190195 200                                                                 GlyValArgTyrTyrSerTrpSerGlyThrSerProLeuThrAsnLeu                              205210215                                                                     LeuAspProSerAspLeuLeuMetGlyAlaSerSerLeuThrPheGly                               220225230                                                                    SerGluAlaAsnGluProGlyArgProLeuGlnPheAlaHisGlyPro                              235240245                                                                     ValIleArgAspAsnTyr ArgMetAsnHisLeuAspGluValAsnGln                             250255260                                                                     ThrLeuGlyLeuThrSerLeuPheGluThrAspProValThrValTyr                              265270275 280                                                                 ArgGlnHisAlaAsnArgLeuLysAsnAlaGlyLeu                                          285290                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AATTCTATGTTCTGGTTCGGCTCGAGCGGCTATACCCAGACCAAATACCCCATCGTCCTA60                GGCCACGGCATGCTGGGTTTCGACAGCATCCTCGGCG97                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 97 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TCGACGCCGAGGATGCTGTCGAAACCCAGCATGCCGTGGCCTAGGACGATGGGGTATTTG60                GTCTGGGTATAGCCGCTCGAGCCGAACCAGAACAT AG97                                      (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GAGCCGAACCAGAAAGCCGCGTGCGC 26                                                 (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CTCGAGCCGAACCAAGCCGCGTGCGC 26                                             

We claim:
 1. Process for preparation of a cholesterol esterase,comprising mutagenizing a gene which codes for a first cholesterolesterase with an active site having amino acid sequence:

    Gly-His-Ser-X-Gly

to change X to an amino acid which differs from the amino acid in saidfirst cholesterol esterase, to form a gene which codes for a secondcholesterol esterase, wherein said second cholesterol esterase hassubstrate specificity different from said first cholesterol esterase,transforming a microorganism with said gene, and culturing saidmicroorganism to produce said cholesterol esterase with differentsubstrate specificity, said mutagenizing said gene being such that X ischanged to His.
 2. Process of claim 1, wherein said mutagenizingcomprises removing an oligonucleotide sequence from said gene andreplacing it by a second oligonucleotide sequence.
 3. Process of claim2, wherein said second oligonucleotide sequence is synthesized viachemical means.
 4. Process of claim 2, wherein said secondoligonucleotide sequence is synthesized via solid phase synthesis. 5.Process of claim 2, wherein said second oligonucleotide sequence issynthesized via a phosphoramidite process.
 6. Process of claim 2,comprising ligating said second oligonucleotide sequence into said gene.7. Process of claim 1, wherein said microorganism does not expressactive cholesterol esterase prior to transformation.
 8. Process of claim1, wherein said microorganism produces cholesterol esterase, and saidprocess further comprises mutagenizing said microorganism so it does notproduce cholesterol esterase prior to transforming said microorganismwith said gene.
 9. Process of claim 8, wherein said microorganism is E.coli or Pseudomonas spec.
 10. Process of claim 9, wherein saidmicroorganism is Pseudomonas spec DSM
 5902. 11. Process of claim 1,comprising introducing said mutagenized gene into said microorganism viaa vector.
 12. Process of claim 11, wherein said vector is pBR322 orpBT306.1.
 13. Non naturally occurring cholesterol esterase producedaccording to the process of claim 1.